The goal of this exploratory project is to investigate the novel hypothesis that increased blood vessel stiffness, which accompanies aging and atherosclerosis progression, contribute to endothelial cell dysfunction. It is now well-known that in vivo, the intimal and medial layers of aged and atherosclerotic vessels tend to be less compliant than younger, healthy vessels. Moreover, it has been shown that arterial elasticity is an independent predictor of cardiovascular mortality in patients over 70 years of age, and those with hypertension and diabetes mellitus. While the macro-mechanical properties of the vessel are well characterized, little is known about how increased matrix stiffness affects the cells within the vessel wall. Specifically, it is unknown whether vessel wall stiffness could actively promote disease progression at the cellular level. We will fabricate engineered substrates that are tuned to mimic the stiffness of healthy and atherosclerotic vessels to investigate endothelial cell-specific responses to matrix stiffness. This project is organized around three specific aims. Aim 1: Determine the effects of matrix elasticity on flow-mediated endothelial cell morphological changes, cytoskeletal rearrangement, integrin adhesion and permeability. In this aim, we will investigate endothelial cell realignment and shape, cytoskeletal rearrangement, integrin activation and monolayer permeability on varying stiffness substrates to test the hypothesis that changes in substrate mechanics alters endothelial cell morphology, focal adhesion formation, and junctional integrity. Aim 2: Investigate the effect of matrix elasticity on ICAM-1 and E-selectin expression and localization and monocyte adhesion in normal and disturbed flow conditions. In this aim, we will test the hypothesis that stiffer matrices, mimicking those of atherosclerotic vessels, will augment the response of endothelial cells to the inflammatory cytokine, TNF-a, causing increased expression of ICAM-1 and E-selectin, increased monocyte adhesion, and increased permeability. Aim 3: To investigate endothelial cell junctional structure and cell orientation in vivo in mice as a function of age and vessel stiffness. In this aim, we will use two-photon microscopy, a state-of-the-art imaging modality that can image several millimeters deep into tissue, to visualize the 3D localization of endothelial cell junctional proteins and endothelial cell morphology in intact arteries as a function of age. Because two-photon microscopy restricts excitation to a femtoliter focal volume and has the ability to penetrate into tissues without dissection of the tissue, we will be able to precisely image junctional structures without the confounding effects of out-of- plane fluorescence and with only minimal manipulation of the vessel. This aim will provide the first 3D reconstructions of the endothelium of intact arteries as a function of age. Moreover, it will validate the results we obtain in Aims 1 and 2 in an in vivo model. Together, this work will elucidate how changes in the stiffness of the basement membrane of the endothelium affects endothelial cell function, with the goal of identifying novel therapeutic targets to prevent the cells' response to vessel stiffness. PUBLIC HEALTH RELEVANCE: During aging and atherosclerosis progression, blood vessels stiffen. While this stiffening has been of intense interest at the organ level, because it results in increased load on the heart, the effect of increased wall stiffness at the cellular level has largely been ignored. This proposal will investigate the effects of increased vessel stiffness on the endothelium, the first mediator in the development of atherosclerosis, in order to identify treatments to prevent aberrant cell behavior and the progression of atherosclerosis.